Human-derived gut microbiota modulates colonic secretion in mice by regulating 5-HT3 receptor expression via acetate production

Abstract

Serotonin [5-hydroxytryptamine (5-HT)], an important neurotransmitter and a paracrine messenger in the gastrointestinal tract, regulates intestinal secretion by its action primarily on 5-HT₃ and 5-HT₄ receptors. Recent studies highlight the role of gut microbiota in 5-HT biosynthesis. In this study, we determine whether human-derived gut microbiota affects host secretory response to 5-HT and 5-HT receptor expression. We used proximal colonic mucosa-submucosa preparation from age-matched Swiss Webster germ-free (GF) and humanized (HM; ex-GF colonized with human gut microbiota) mice. 5-HT evoked a significantly greater increase in short-circuit current (ΔI_sc) in GF compared with HM mice. Additionally, 5-HT₃ receptor mRNA and protein expression was significantly higher in GF compared with HM mice. Ondansetron, a 5-HT₃ receptor antagonist, inhibited 5-HT-evoked ΔI_sc in GF mice but not in HM mice. Furthermore, a 5-HT₃ receptor-selective agonist, 2-methyl-5-hydroxytryptamine hydrochloride, evoked a significantly higher ΔI_sc in GF compared with HM mice. Immunohistochemistry in 5-HT_3A-green fluorescent protein mice localized 5-HT₃ receptor expression to enterochromaffin cells in addition to nerve fibers. The significant difference in 5-HT-evoked ΔI_sc between GF and HM mice persisted in the presence of tetrodotoxin (TTX) but was lost after ondansetron application in the presence of TTX. Application of acetate (10 mM) significantly lowered 5-HT₃ receptor mRNA in GF mouse colonoids. We conclude that host secretory response to 5-HT may be modulated by gut microbiota regulation of 5-HT₃ receptor expression via acetate production. Epithelial 5-HT₃ receptor may function as a mediator of gut microbiota-driven change in intestinal secretion.NEW & NOTEWORTHY We found that gut microbiota alters serotonin (5-HT)-evoked intestinal secretion in a 5-HT₃ receptor-dependent mechanism and gut microbiota metabolite acetate alters 5-HT₃ receptor expression in colonoids.View this article's corresponding video summary at https://www.youtube.com/watch?v=aOMYJMuLTcw&feature=youtu.be.

Keywords: 5-hydroxytryptamine type 3; irritable bowel syndrome; microbiome; motility; physiology; secretion.

Fig. 1.

Microbial colonization does not affect intestinal permeability and baseline secretion. TER (A), baseline I_sc (B), and forskolin (10 µM)-evoked ΔI_sc (C) in GF and HM mice.

Fig. 2.

The maximal secretory response to 5-HT is greater in GF than HM mice. A: change in short-circuit current in response to cumulative concentration of 5-HT in HM and GF mice. B: 5-HT-induced E_max in GF and HM mice. *P < 0.05.

Fig. 3.

5-HT_3B receptor mRNA expression is higher in GF mice than HM mice. A: relative expression of 5-HT_3B mRNA in HM and GF mice. B: relative expression of 5-HT₄ mRNA in HM and GF mice. *P < 0.05.

Fig. 4.

5-HT_3B receptor protein expression is higher in GF mice than HM mice. A: 5-HT_3B subunit antibody detected two bands at ~50 and 40 kDa. Controls include blockade of antibody binding by preincubating the primary antibody with its competing peptide (1°+B.P/2°) and secondary-only controls (2° Only). B: representative blot of 5-HT_3B subunit and GAPDH expression in GF and HM mouse proximal colon. C and D: quantification of the 5-HT_3B subunit band intensity present at 50 kDa (top band) and 40 kDa (bottom band), respectively, in HM and GF mice. *P < 0.05.

Fig. 5.

5-HT₃ receptor antagonist inhibits 5-HT-evoked ΔI_sc only in GF mice whereas 5-HT₄ receptor antagonist significantly blocks ΔI_sc in both HM and GF mice. A: change in short-circuit current in response to cumulative concentration of 5-HT and 5-HT in the presence of ondansetron in GF mice. B: change in short-circuit current in response to cumulative concentration of 5-HT and 5-HT in the presence of ondansetron in HM mice. C: change in short-circuit current in response to cumulative concentration of 5-HT and 5-HT in the presence of ondansetron in CR mice. D: change in short-circuit current in response to cumulative concentration of 5-HT and 5-HT in the presence of GR-113808 in GF mice. E: change in short-circuit current in response to cumulative concentration of 5-HT and 5-HT in the presence of GR-113808 in HM mice. F: change in short-circuit current in response to cumulative concentration of 5-HT and 5-HT in the presence of GR-113808 in CR mice. 5-HT-induced E_max for GF (G), HM (H), and CR (I) mice in the presence of ondansetron (Ond) and GR-113808 (GR). *P < 0.05.

Fig. 6.

Increased secretory response to 5-HT in GF mice is mediated by 5-HT₃ receptor. 5-HT₃ receptor-selective agonist shows a significantly greater secretory response in GF mice compared with HM mice. *P < 0.05.

Fig. 7.

5-HT₃ receptors are expressed in the epithelial cells of colonic mucosa and in submucosal neuronal fibers. A: arrows highlight 5-HT_3A-GFP-positive cells in the mouse colonic epithelium. A subset of 5-HT₃-GFP-positive cells (B) colocalize with 5-HT and DAPI (C) in the proximal colon.

Fig. 8.

Epithelial 5-HT₃ receptor is responsible in part for the differential secretory response to 5-HT. Change in short-circuit current in response to cumulative concentration of 5-HT in the presence of TTX (A), change in short-circuit current in response to cumulative concentration of 5-HT in the presence of TTX and ondansetron (B), and change in short-circuit current in response to cumulative concentration of 5-HT in the presence of TTX and GR-113808 (C) in GF and HM mice. *P < 0.05.

Fig. 9.

Incubation of GF colonoids with acetate significantly decreases 5-HT_3B receptor mRNA expression. A: effect of acetate on 5-HT_3B receptor expression in GF colonoids. B: effect of butyrate on 5-HT_3B receptor expression in GF colonoids. *P < 0.05.